Basement membranes are complex extracellular matrix structures that are essential for the development, organization, and maintenance of tissues and complex organisms. They are in intimate contact with cell surfaces and thereby influence cell behavior by mechanisms that are not fully understood. Several well-defined macromolecular components such as type IV collagen, laminin, heparan sulfate proteoglycan and entactin have been localized to the basement membrane. The biosynthesis and assembly of this supra-molecular matrix and the mechanisms which regulate them in normal and diseased states require further study. The objective of this proposal is to address those problems. The specific aims of the proposal are: I. To analyze the regulation of assembly of the A, B1 and B2 chains of laminin; II. To study the assembly of the laminin-entactin complex. To accomplish these objectives the assembly process will be studied in both intact cells and cell-free systems. Antibodies specific for each molecule will be obtained for the analyses. Vectors containing cDNA sequences for each of the laminin chains will be constructed and used to perturb the intracellular assembly of laminin. Stable cell lines which produce modified matrices will be obtained and studied. The interaction of laminin and entactin may play an important role in regulating basement membrane function and specificity. Entactin will be produced in the baculovirus expression system and its re- constitution with laminin will be examined. In addition, the intracellular assembly of the laminin-entactin complex will be studied by modulating it by transfection with vector constructs containing entactin cDNA sequences. The ultimate goal of this work is to understand how basement membranes are assembled, what regulates the assembly, and how the specificity of basement membrane function in health and disease is related to these processes.